Calcitriol Revises Aromatase Gene Expression in Ehrlich Solid Tumor Bearing Mice Exposed to Low Dose Gamma Radiation

Research Article | DOI: https://doi.org/10.31579/2640-1053/074

Calcitriol Revises Aromatase Gene Expression in Ehrlich Solid Tumor Bearing Mice Exposed to Low Dose Gamma Radiation

  • Nermeen M El Bakary 1*
  • Khaled Sh Azab 1
  • Shaden M. Hanafy 2
  • Ghada M Abdel Aziz 2
  • Aysam M. Fayed 2

1* Radiation Biology Department, Radiation Research Division, National center for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt.
2 Molecular biologyDepartment, Institute of Genetic Engineering and Biotechnology, University of Sadat City, Egypt.

*Corresponding Author: Nermeen M El Bakary, Radiation Biology Department, Radiation Research Division, National center for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt.

Citation: N M El Bakary, Kh Sh Azab, S M. Hanafy, G M Abdul Aziz and A M. Fayed. (2021) Calcitriol Revises Aromatase Gene Expression in Ehrlich Solid Tumor Bearing Mice Exposed to Low Dose Gamma Radiation. Cancer Research and Cellular Therapeutics. 5(1); Doi: 10.31579/2640-1053/074

Copyright: © Nermeen M El Bakary, This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium,provided the original author and source are credited.

Received: 26 February 2021 | Accepted: 15 March 2021 | Published: 17 March 2021

Keywords: calcitriol; gamma radiation; aromatase; cox2; pge2; caspase3; tumor volume

Abstract

Background: Dysregulation of aromatase expression had been monitored in many types of cancer. Our study aimed to evaluate the possible role of calcitriol (Cal; Vit D3-OH) or/and low dose of gamma radiation in regulation of aromatase gene expression and the regression of tumor proliferation in murine model (EST; Ehrlich solid tumor bearing mice).

Methods: Mice with ≈1 cm3 EST were received (i.p. injection) day after day repeated doses of Calcitriol (Cal) (0.05µg/mouse) for 14day or/and exposed to 0.5 Gy gamma radiation (low dose) delivered as one shot at dose rate 0.48 Gy/min.

Results: Our results demonstrated that, mRNA expression of aromatase, levels of cyclooxygenase (COX2) and prostaglandin (PGE2) in addition to volume of the tumor are significantly decreased while caspase 3 level is significantly increased in EST mice treated with Cal or/and exposed to 0.5 Gy gamma ray compared to untreated EST bearing mice. However, the most pronounced improvements in all of the measured parameters were obviously indicated in EST mice group treated with Cal and exposed to gamma radiation. This was accomplished by suppression of inflammatory markers which cause down regulation in aromatase mRNA expression as well as augmenting apoptosis by inducing Caspase3 concentration.

Conclusion: It could be concluded that the exposure to low dose gamma radiation potentiate the action of Calcitriol against tumor growth in the subjected murine model which represent a prospective policy for the management of solid tumor and decreasing the possibilities of tumor drug resistance. 

Introduction

The medical history of cancer began millennia ago. Historical findings of patients with cancer date back to ancient Egyptian and Greek civilizations, where this disease was predominantly treated with radical surgery and cautery that were often ineffective, leading to the death of patients.[1] Searching for novel approaches to control malignancies is the noble target of scientists working in the field of cancer research and many other related fields. Currently, several researchers are focused on the development of cell therapies, anti-tumor vaccines, and new biotechnological drugs that have already shown promising results in preclinical studies, therefore, in the near future, we will certainly assist to a new revolution in the field of medical oncology [2, 3, 4].

For many years, breast cancer has had the highest incidence of all cancers in women worldwide. There are significant data implicating estrogen and its metabolites in the development of breast cancer. Estrogen binding to its receptor results in proliferation of estrogen-sensitive epithelial tissues. With rapid proliferation, there is the potential for genetic errors and consequent predisposition to malignant cellular transformation [5]. Alternatively, estrogen metabolites may have intrinsic genotoxic effects and cause DNA damage and cellular transformation.6 Postmenopausal combined hormone therapy increases the risk of breast cancer, the chances of dying from breast cancer, and the likelihood that the cancer may be found only at a more advanced stage. There are many evidences linking between estrogens, largely contributing to breast cancer development and other types of cancer [7,8,9]. Estrogen activates tumor growth and inhibits apoptosis through mechanisms mediated by its interaction with estrogen receptors [10].

Aromatase (CYP19), a cytochrome P450 enzyme, responsible for estrogens biosynthesis is important in the growth of breast cancers in both pre- and postmenopausal women. It is highly expressed in human breast cancer tissue compared to normal breast tissue. Further, in situ estrogen production is more effective than circulating estrogen in the promotion of breast cancer.  The tumor aromatase has been shown to stimulate breast cancer growth via an autocrine and a paracrine manner [11]. Estrogen synthesized locally becomes the major source of the hormone in the breast after menopause when circulating estrogen levels from the ovaries dramatically decline [12].

As aromatase catalyzes the final and rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme are effective targeted therapy.  The aromatase inhibitors (AIs), a standard treatment for postmenopausal patients are effective in adjuvant and first-line metastatic setting. Aromatase inhibitors lower estrogen levels by stopping aromatase from changing other hormones into estrogen (Estrogen can fuel the growth of breast cancer cells). Preclinical data in resistance models demonstrated that the crosstalk between estrogen and other signaling pathways is an important resistant mechanism. Blockade of these signaling pathways is an attractive strategy to overcome the resistance to AI. There are several clinical trials to evaluate novel targeted therapies to reverse resistance to AIs [13,14,15].

We are interested in Vit D3 (Cholecalciferol) which is derived from food and is essential precursor to the potent steroid hormone, 25- dihydroxy vitamin D3, 1α,25(OH)2D3, or calcitriol. Hormonally active Vit D (calcitriol) has been claimed for its critical importance in the case of health or disease. The decrease in vitamin D content leads to group of abnormalities advanced to serious diseases whereas, the over consumption of Vit D leads to several undesired symptoms [16]. The inhibitory action of calcitriol on aromatase expression has been subjected to investigation in our present study.

Evidences have been accumulated to indicate that calcitriol modulates calcium and bone metabolism. These include anti-proliferation, anti-angiogenesis, pro-apoptosis, pro differentiation, and immune regulation in a cell and tissue [17].Also, the calcitriol signaling plays an important role in the development of normal mammary gland [18].

Studies on vitamin D receptor (VDR) knockout mice provide evidence that calcitriol signaling through the VDR opposes estrogen driven proliferation of mammary epithelial cells and maintains normal differentiation [19].

Low dose gamma irradiation has been authenticated as a promising auxiliary strategy to improve the immune adaptive response and its implication during the strategy of cancer treatment indicates many intriguing results [3, 4, 20].

Feinendegen reported that radiation hormesis is the hypothesis that low doses of ionizing radiation (A low dose of IR is a dose that creates a burst of hits and ROS that is adequate to stimulate the protective systems and produce observable health benefits, within the region of and just above natural background levels in the range 0.01-0.50 Gy  )[21] are beneficial, stimulating the activation of repair mechanisms that protect against disease, that are not activated in absence of ionizing radiation [22].Hence, the radiation adaptive response direct DNA repair and gives the impression to be a central origin of the prospective hormetic effect [23].

The pro inflammatory molecules; prostaglandins (PGs) and its synthesis rate limiting enzyme, cyclooxygenase-2 (COX2) play an important role in the development and progression of cancer [24].Many acknowledged evidences addressed the correlation between over expression of Aromatase and expression of inflammatory molecules (PGS and COX2) [25,26].

The irradiation of EST bearing mice at low dose was taken as argue of immune system [27,28]. this work explored the role of vitamin D3 analogue; Calcitriol as an Aromatase  inhibitor  in regulating the cancer proliferation in mice treated with low dose and to what extent it can reduce the possibility of cancer treatment failure in  Ehrlich solid tumor (EST) bearing mice exposed to low radiation dose.

Material and Methods

Animals

Female swiss albino mice (1 years old) weighing (30-35g) were obtained from the National Cancer Institute (Cairo University, Giza, Egypt). Animals were housed in plastic cages and maintained under standard conditions of temperature, humidity and 12 h light/dark cycles along the experimental period. They were provided with a pellet diet containing all the necessary nutritive elements. Food and water were available throughout the experimental periods ad libitum. Mice were left for acclimatization period of 1 week before the start of the experiment.

Animal experimentation were consistent with the guidelines of Ethics by the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) in accordance with the recommendations for the proper care and use of laboratory animals approved by Animal care committee of the National Center for Radiation Research and Technology (NCRRT), Cairo, Egypt.

Calcitriol (Cal)

Calcitriol (Vitamin D3 capsule; 1000 IU) was obtained from Puritan's Pride Company (Cairo, Egypt). It was dissolved and diluted (1:10 v/v) in saline. Each mouse received intraperitoneal (i.p) injection of 0.05µg/mouse once every 48 h for 14 days [29].

Tumor Inoculation

The initial inoculums of Ehrlich ascites carcinoma (EAC) cells, kindly provided by the National Cancer Institute, Cairo University, and Giza, Egypt) were propagated in our laboratory. This was performed by weekly i. p. injection of freshly ascites fluid withdrawn from a donor mouse (bearing 6-8 days old ascites tumor) into 4 female mice each weighing 35g approximately using sterile disposable syringes under aseptic conditions. Whereas, the cell line of EAC was preserved by ip injection of 2.5 million cells per animal. To count the EAC before ip, the bright line hemocytometer was used. Physiological sterile saline solution was used for the injection and dilution. Solid tumor of Ehrlich carcinoma was obtained by the intramuscular transplantation of 2.5 x106 viable Ehrlich cells into the right thigh muscle of the lower limb of healthy mice, and obtained after two week of inoculation ( zero time of the experiment)[3]

Tumor Volume Monitoring

Tumor volume was measured at different time intervals during the experimental period using a Vernier caliper on the 15th, 21th, and 28st days from the time the tumor reached 1 cm3 during the experimental period. The volume of solid tumor was calculated using the following formula according to Papadopoulos ET al [30]: [A × B2× 0:52], where A and B are the longest and the shortest diameter of tumor, respectively

Low Dose Gamma Irradiation

Whole body gamma irradiation to mice was performed using   a Gamma cell 40 Cesium Irradiator (Atomic Energy of Canada Ltd., Ontario, Canada) located in the National Center for Radiation Research and Technology (NCRRT) of the Atomic Energy Authority (EAEA,Cairo, Egypt) which ensured a homogenous dose distribution all over the irradiation tray.  Mice were placed in a specially designed well-ventilated acrylic container and each mouse was whole body irradiated at 0.5Gy, [31] at a dose rate of 0.48Gy/min calculated by the authorization of the Dosimeter Department in the NCRRT.

Animal grouping

A total number of 90 female Swiss albino mice were randomly allocated into 6 equally- sized groups as follows:

Group 1 (Positive control, E):  Mice bearing solid Ehrlich tumor

Group 2 (Irradiated, R): Mice of this group were whole body exposed to 0.5 Gy of γ-radiation at the day 15 of the experiment.

Group3 (Calcitriol, Cal): Mice of this group were intraperitoneally injected with Cal (0.05 µg Cal /mouse) every other day for 14 day starting at 14 days from initiation of the experiment (from zero time).

Group 4 (E+ R): Mice of this group are bearing solid tumor and then exposed to 0.5 Gy of whole body γ-radiation.

Group 5 (E+ Cal): Mice of this group are intraperitoneally injected with 0.05 µg Cal/mouse once every other day for 14 days starting on the 15th day from experiment.

Group 6 (E+ R+ Cal): Mice of this group are exposed to 0.5 Gy whole body γ-irradiation and intraperitoneally injected with 0.05 µg Cal/mouse once every other day for 14 days starting on the 15thday from the day of Ehrlich cells inoculation.

Samples preparation

At the day after complete all treatments according to the experiment suggested regimen (15th days from the experiment zero time); mice were anesthetized using diethyl ether. The blood was collected on heparinized tubes by using syringe for heart puncture and the collected blood were preserved at -80 ºC for the molecular analysis (RT-PCR). Animals were sacrificed and undergo necropsy. Portions of solid tumor were dissected, rinsed in ice cold isotonic saline, blotted dry with a filter paper and preserved at -20 ºC for ongoing biochemical analysis.

RT-qPCR assessment of Aromatase gene expression

Entire cellular RNA was extracted from the thigh muscles by use of RNeasy® Mini Kit (Qiagen, Hilden, Germany). The procedures were done according to the manufacturer’s instructions. The total RNA concentration and also the purity of RNA samples were determined by using the Nano Drop spectrophotometer (Thermo Fisher Scientific, USA). Purity of samples was determined by measuring absorbance at 260 and 280 nm, respectively.

First-strand complementary DNA (cDNA) was synthesized using Thermo Scientific™ Revert Aid™ First-Strand cDNA Synthesis Kit (Fermantas,   Thermo Fisher Scientific Inc, Runcorn, UK).

Real-time polymerase chain reaction (PCR) amplification and analysis were performed in an optical 96-well plate in ABI PRISM 7500 Fast Sequence Detection System Thermal Cycler (Applied Biosystems, Foster City, CA, USA) using Power SYBRR Green PCR Master Mix (Applied Biosystems).

The relative expression of the real-time reverse transcriptase PCR products was determined by the ΔΔCt method. This method calculates a relative expression to housekeeping gene using the equation: fold induction = 2^ - (ΔΔCt). Where ΔΔ Ct=Ct [gene of interest (unknown sample)-Ct housekeeping gene (unknown sample)] - [Ct gene of interest (calibrator sample) - Ct housekeeping gene (calibrator sample)] [32].

Primers used for aromatase RNA gene expression and glyceraldehyde -3- phosphate dehydrogenase (GAPDH) as a housekeeping gene are as follow

Table 1:  Sequences of primers used to accomplish RT-PCR amplification

Biochemical Analysis

Caspase 3 was assayed in solid EC tumor by a sandwich enzyme-linked immunosorbent assay (ELISA) mouse kit provided by R&D Systems, Inc. (Minneapolis, USA according to the kits manufacture procedures. Whereas, Prostaglandin level in solid EC tumor was determined by a sandwich enzyme-linked immunosorbent assay (ELISA) mouse kit provided by Enzo life science (Postfach, Switzerland). The COX-2 enzyme activities were detected spectrophotometrically in tissue homogenate. Cyclooxygenases catalyze the synthesis of prostaglandins from arachidonic acid. The COX component converts arachidonic acid to Prostaglandin G2 (PGG2) whereas the peroxidase component reduces the PGG2 to the corresponding alcohol, Prostaglandin H2 (PGH2). In this kit the TMPD serves as a reducing agent, TMPD gets oxidized, so electrons flow from the TMPD to PGG2 and the appearance of the oxidized TMPD is monitored at 590 nm. The rate of increase in absorbance was taken for calculating the activity of the enzyme [33].

Statistical analysis

All data were subjected to the Shapiro–Wilk test for normality (p >0.05) to ensure the normal distribution. After confirmation of data normal distribution statistical analysis of the difference between means was performed using one-way analysis of variance (ANOVA). When significant F-ratio was present, the post hoc Duncan’s test for multiple comparisons was used to evaluate the statistical significance difference between various groups (p < 0>

Results

mRNA expression of Aromatase

The present study comprises the assessment of Aromatase gene expression using RT-PCR technique. Table (2) Shows that the highest mRNA expression of Aromatase was found in the Ehrlich solid tumor of E mice group which considered as +ve control group whereas, the lowest level was recorded in group 3 (Cal) mice. The results demonstrated the most expression of Aromatase mRNA in untreated solid tumor considered as 100% expression and the administration of several equal doses of Cal reduced the expression to 78.3%.Whereas, the exposure of Ehrlich bearing mice to single dose of 0.5Gy reduced to 76% compared to expression of that in the untreated Ehrlich bearing mice. The most pronounced reduction amongst groups was observed in E+ R+ Cal.

Table 2: Comparative mRNA [removed]arbitrary units) of Aromatase in the right thigh muscle or solid tumor of different animal groups

R: Radiation     E: Ehrlich       Cal: calcitriol

Each value represents Mean± SE (n=15)

Values with dissimilar superscript letters are considered significantly different (p <0>

%: Percentage of change from the values of mRNA in mice bearing Ehrlich solid tumor (E).

Percent change = T-C/C* 100

Inflammatory markers

COX2

The protein concentration of COX2 in different animal groups showed a great variation as shown in Table (3).

The result illustrated a marked down regulation of COX- 2 protein in (E+R+Cal) gp of mice in comparison to (E+R) gp or (E+Cal) gp.

Table 3: Cyclooxygenase-2 (COX-2) concentration in different animal groups

R: Radiation     E: Ehrlich       Cal: calcitriol

Each value represents Mean± SE (n=15)

Values with dissimilar superscript letters are considered significantly different (p <0>

%: Percentage of change from the values in mice bearing Ehrlich solid tumor (E).

Percent change = T-C/C* 100

PGE2

A significant increase of tumor PGE2 concentration in (E) gp of mice and (E+R) gp of mice compared to the other gps as shown in table (4)

In mice bearing solid tumor and pretreated with Cal a remarkable (p<0>

Table 4: Prostaglandin E2 (PGE2) level in different animal groups

R: Radiation     E: Ehrlich       Cal: calcitriol

Each value represents Mean± SE (n=15)

Values with dissimilar superscript letters are considered significantly different (p <0>

%: Percentage of change from the values in mice bearing Ehrlich solid tumor (E).

Percent change = T-C/C* 100

Apoptosis

The experimental data relating to the activities of caspase3 in different animals’ groups is presented in Table (5).

Table shows that E mice manifested a significant (p<0>

Treatment of E mice with Cal resulted in a significant elevation in caspase-3 concentration, compared to E mice. Further, exposure of E mice to γ-irradiation either alone or in combination with Cal treatment induces significant (p<0>

Table 5: Caspase-3 protein expression in different animal groups

R: Radiation     E: Ehrlich       Cal: calcitriol

Each value represents Mean± SE (n=15)

Values with dissimilar superscript letters are considered significantly different (p <0>

%: Percentage of change from the values in mice bearing Ehrlich solid tumor (E),

Percent change = T-C/C* 100

Tumor proliferation

The volume of solid tumor in the right thigh of mice was measured three times starting after pulp appearance in E mice. The data obtained has been represented in Table (6) and figure (1). Table displays the effect of Calcitriol (Cal), Gamma irradiation (R) and their combination on the growth of solid tumor throughout 28 days’ post EAC inoculation. Treatment of animals with Cal starting after the tumor volume reached 1 cm3 (14 days after tumor transplantation) resulted in marked inhibition of tumor growth as observed at 15, 21 and 28 days from the day of EAC inoculation. The exposure of EAC-bearing mice (E) to γ-irradiation resulted in a considerable reduction in the tumor size. Also, significant reduction of tumor volume was identified in irradiated mice bearing tumor and treated with Cal (E+ R+ Cal mice group) at 15, 21 and 28 days from the day of EAC inoculation compared to that E mice. It worth mention that, the (E +R+ Cal) mice signify the most reduced tumor volume as compared with other groups.

Table 6: Influence of Cal administration or/and exposure to 0.5 Gy gamma radiation on Tumor growth (mm3) in different animal

R: Radiation     E: Ehrlich       Cal: calcitriol

Each value represents Mean± SE (n=15)

Values with dissimilar superscript letters are considered significantly different (p <0>

Figure 1: Influence of Cal administration or/and exposure to 0.5 Gy gamma radiation on Tumor growth (mm3) in different animal

R: Radiation     E: Ehrlich       Cal: calcitriol

Each value represents Mean± SE (n=15)

Values with dissimilar superscript letters are considered significantly different (p <0>

Discussion

The development of original approaches in cancer treatment to arrive new level of success in the continuous battle against cancer required searching deeply in the relative tiny details. One of the interesting tricky points is the relation between estrogens and breast cancer. Our study focused on the aromatase enzyme a rate limiting enzyme in the pathway of estrogen synthesis. Likewise, the present work comprises the examination of calcitriol (vit D3-OH) or/and low dose gamma irradiation as novel approaches targeting the adjustment of aromatase expression, reducing the tumor proliferation and overcoming the disadvantage of using ordinary aromatase inhibitors.

While, aromatase (CYP19) catalyzes the synthesis of estrogens from androgenic precursors and ovaries are the principal source of circulating estrogens in premenopausal women, many other tissues including the breast express aromatase and hence have the capacity to locally synthesize estrogens [34].Tissue specific promoters differentially regulated by various transcription factors are controlled the aromatase expression in these different situates [35].Lønning et al. stated that circulating pool of estrogens may be more important for breast carcinogenesis than local estrogen synthesis upregulated in postmenopausal benign breast tissue or malignant breast tumors [36].

In the present study, the data obtained pointed out to significantly higher (p<0>

Worth mention, mouse model is not being an exact mirror of estrogen production in humans since mouse aromatase is expressed in fewer tissues than human aromatase [38]. However; this was going over in our work as a highly expressed aromatase gene was observed in solid tumor of mice inoculated with EAC cells (Table 2). The upregulation of aromatase gene expression could be attributed to the presence of multiple exons available for use in aromatase gene.The regulation of tissue specific aromatase gene by alternative uses of these exons has been identified [39]. During cancer development exon 1 termed exon I.4 is often toggled from exon I.4 (1b) to exon I.3 (1c) or exon PII (1d), (1b) which causes enhancement of aromatase expression in cancer-associated adipocytes and fibroblasts. Moreover, the aromatase gene is further regulated at both the transcriptional and post-transcriptional levels through protein kinase A (PKA)-, protein kinase C (PKC)-, and tyrosine kinase receptor-mediated signaling pathways that employ prostaglandin E2 and class 1 cytokines. Epigenetic modifications of the aromatase gene and microRNA mediated aromatase regulation may play a critical role in cancer progression. Several genetic polymorphisms in the aromatase gene may be prognostic factors of disease and may influence response to aromatase inhibitors.

Lately, inhibition of aromatase has become one of the key strategies in breast cancer treatment. In the present work, we recorded a significant decrease in the expression of aromatase in EST mice treated with calcitriol or/and exposed to low dose gamma irradiation (LDR) with remarkable advantage of mice received Cal and exposed to LDR (Table 2). The results obtained signalize obviously the capability of calcitriol to reduce aromatase gene expression even applied lonely or with LDR. As an inception, calcitriol could exert anti proliferative, pro-apoptotic and pro-differentiation actions in several malignant cells including   breast cancer cells. Thoroughly, it can inhibit the expression of CYP19A1 gene encoding aromatase enzyme that catalyzes estrogen synthesis from androgenic precursors. In addition, calcitriol repress nuclear receptor responsible for estrogen actions; Era [40]. These mutual actions (opposing the estrogen effect and inhibition of aromatase mRNA expression) trim down the levels of the estrogenic hormones as well as their biological activities within the breast [41]. It was previously mentioned the presence of different promoters distributed over a regulatory region of the CYP19A1 gene permit regulation of the aromatase gene in a tissue selective manner [42]. The main beneficial action of calcitriol is its ability to selectively diminished aromatase transcription. Promoters responsible for aromatase gene expression not only differ between normal tissues but also between normal and   cancer tissue. So, tissue-selective CYP19A1 promoters have been proposed as a possible strategy for development of selective aromatase modulators (SAM) [43]. Wheler et al. mentioned that AIs, a main therapeutic approach to treat breast cancer, prevent progression or recurrence of cancer breast in postmenopausal women after primary therapy have a serious disadvantageous of inhibiting estrogen synthesis at any sites as bone [44,45].

Whereas, calcitriol could inhibit the production of estrogen receptors as well as suppress other growth signaling pathway selectively in tumor cells [46]. Furthermore, the improvement recorded in aromatase expression in mice exposed to low dose gamma radiation (Table2) could be due to enhancement of the immune responses because there are no consequential evidences of direct effect of low level of gamma radiation on the machinery of aromatase gene expression. However, LDR may   potentiate the action of Cal on the unique exon’s arrangement in the structure of aromatase gene.

Just about the same issue, our experimental results identify significant increases in the protein expression of COX2 and PGE2 synthesis synchronous with highly expressed aromatase mRNA (Tables 2:4). We hypothesized the correlation between inflammatory response and aromatase gene expression in the development of cancer. We expected the contribution of Inflammation/ Aromatase axis to the increased risk of hormonal developing cancer. One could attribute the changes induced in COX 2 and PGE2 protein to the inflammatory burden implicated after inoculation of EAC cells in the right thigh of normal mice which in turn work to enhance aromatase expression. In agreement with this view Grivennikov et al. reported that the inflammatory responses play influential roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion, and metastasis [47]. The most, if not all, solid malignancies trigger an intrinsic inflammatory response that builds up a pro tumorigenic micro environment [35]. Tak and Firestein, nominate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) as a link between inflammation, immunity to cancer development and progression [48]. NF-κB, highly activated at sites of inflammation in diverse diseases, can induce transcription of pro inflammatory cytokines, chemokines, adhesion molecules, matrix metalloproteinases (MMPs), COX2 [49,50].

Cyclooxygenases COX 1 and COX2 are rate limiting enzymes in the production of PGE2 from arachidonic acid. The major pathophysiological functions of   PGE2 are bringing out pyrexia, pain sensation, and inflammation. So, the analgesic and anesthetic effects of the most widely used non-steroidal anti-inflammatory drugs (NSAIDs) are thought to be driven by inhibition of the production of PGE2. Going to the point, COX-2-derived PGE2 working to stimulates the cyclic adenosine monophosphate (cAMP) to PKA signal transduction pathway that activate CYP19 transcription resulting in increased aromatase expression [51]. Looking upon EST mice received doses of Cal or/and exposed to LDR, significant decreases in the levels of COX2 and the concentration of PGE2 were observed (Table 3 and 4). Calcitriol might play a noticeable role in preventing the exaggeration of inflammation in the tumor tissue of murine mice. Han et al. stated that pro inflammatory cytokines and COX 2 proteins are significant remarks of in situ active inflammatory responses [52]. Meanwhile the decreases happened in the COX2 protein expression and PGE2 concentration in EST mice received calcitriol treatment denotes its anti-inflammatory capacity. It could be suggested that calcitriol barricade the COX2 inducible cytokines in tumor tissue and consequently checked the production of COX2 in tumor tissue. The COX2 enzymatic activity upregulation could be attributed to the activation of several tumor necrosis factor alfa (TNF-α) dependent intracellular signaling pathway which plays a key role in the control of COX-2 induction. Different protein kinases and transcriptional factors that are greatly regulated by TNF-α are contributed to COX2 upregulation. The expression of COX2 mRNA and protein is often enhanced in various human cell types by inflammatory cytokines such as TNF-α [28].As COX2 is the key enzyme in pathway of production of pro inflammatory PGs, synthesis and biological actions of PGs significantly withdrawn [41]. Furthermore, calcitriol could decrease PGE2 via enhancing the expression of 15-Hydroxyprostaglandin dehydrogenase (15-PGDH), the enzyme initiating PG catabolism [53].

Besides, low-dose radiation can be beneficial to living organisms, since the mild oxidative stress induced by low-dose radiation induces adaptive responses, including enhanced cell damage repair/protection [3, 54]. Thus, the decreases in COX2 expression and PGE2 concentration in EST mice exposed to low dose gamma radiation could be interpreted in the view of the hormetic effect of LDR. Further, in EST mice exposed to LDR and received calcitriol demonstrate prevalent reduction of COX2 and PGE2 expression holding up the view of LDR advancing action of calcitriol.

Standing on the previous we have two cohesive points to be followed in concern of calcitriol or and low dose gamma radiation impact against cancer proliferation in EST bearing mice. First which we already discuss is the Over expression of aromatase gene and second is the inhibition of apoptosis in response to highly expressed cycloxygenase2 and PGE2 which we are going to interpret.

The experimental data indicate that calcitriol administration or/and LDR exposure to EST mice induced significant increases in Caspase3 level compared with E mice (Table 5). The reported increases in caspase 3 tailored the promotion of apoptotic process in tumor cells after calcitriol administration. Krishnan et al. affirmed the anticancer effects of Calcitriol due to its ability to suppress proliferation and promotes apoptosis and differentiation of malignant cells and inhibits tumor angiogenesis and invasion [60].

Essentially most pathways which are leading to apoptosis are ultimately regulated by Bcl-2 family of proteins. The Bcl-2 family of proteins is divided into two subclasses that either promote (BclXS, Bax) or suppress (Bcl-2, Bcl-XL, Mcl-1) apoptosis. These proteins can form hetero- and homodimers and the ratio of apoptosis promoters to apoptosis suppressors is one determinant of cellular response. The increases in Bcl-2 expression was observed in a number of cancers including prostate, lung, and breast and is often associated with advanced stages of the disease [61]. Expected upregulation of Bcl2 could be confirmed by the over expression of COX2 and PGE2 (greatly linked toBcL2 prompting) recorded in the present study. On the other hand, the inhibition of this inflammatory signs due to Cal (Table2 and 3) could prevent the up regulation of this pre survival proteins. As Bcl2 protein is critical in preventing the cell from initiating apoptosis, calcitriol have been sought to provide potential anti-cancer therapies by making cancer cells more susceptible to apoptosis-inducing agents.

In addition, the improvement in Caspase 3 expression in EST mice exposed to LDR could interpreted in the view of LDR hormetic effect. The LDR activation of apoptosis could be executed in cancer cells by different mechanisms like inhibition of inflammatory signs, inhibition of aromatase as well as promotion of proapoptotic proteins [3,4]. Xin et al. reported that LDR induces apoptosis of tumor cells and has numerous beneficial effects on normal tissues, including radiation homeostasis and adaptive response [62].

In conclusion Calcitriol and low dose gamma irradiation could introduce transferable potentiating effect towards the cancer proliferation via selective suppression of tumor tissue m-RNA aromatase and amendment of inflammatory response which trigger apoptosis in tumor cells in murine model of EST. This may represent a prospective therapeutic policy for the management of solid tumor and decreasing the possibilities of tumor therapeutic resistance. 

Conflicting interests

The Authors declare that there is no conflict of interest.

References

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Testimony of Journal of Clinical Otorhinolaryngology: work with your Reviews has been a educational and constructive experience. The editorial office were very helpful and supportive. It was a pleasure to contribute to your Journal.

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Pedro Marques Gomes

Dr. Bernard Terkimbi Utoo, I am happy to publish my scientific work in Journal of Women Health Care and Issues (JWHCI). The manuscript submission was seamless and peer review process was top notch. I was amazed that 4 reviewers worked on the manuscript which made it a highly technical, standard and excellent quality paper. I appreciate the format and consideration for the APC as well as the speed of publication. It is my pleasure to continue with this scientific relationship with the esteem JWHCI.

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Bernard Terkimbi Utoo

This is an acknowledgment for peer reviewers, editorial board of Journal of Clinical Research and Reports. They show a lot of consideration for us as publishers for our research article “Evaluation of the different factors associated with side effects of COVID-19 vaccination on medical students, Mutah university, Al-Karak, Jordan”, in a very professional and easy way. This journal is one of outstanding medical journal.

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Prof Sherif W Mansour

Dear Hao Jiang, to Journal of Nutrition and Food Processing We greatly appreciate the efficient, professional and rapid processing of our paper by your team. If there is anything else we should do, please do not hesitate to let us know. On behalf of my co-authors, we would like to express our great appreciation to editor and reviewers.

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Hao Jiang

As an author who has recently published in the journal "Brain and Neurological Disorders". I am delighted to provide a testimonial on the peer review process, editorial office support, and the overall quality of the journal. The peer review process at Brain and Neurological Disorders is rigorous and meticulous, ensuring that only high-quality, evidence-based research is published. The reviewers are experts in their fields, and their comments and suggestions were constructive and helped improve the quality of my manuscript. The review process was timely and efficient, with clear communication from the editorial office at each stage. The support from the editorial office was exceptional throughout the entire process. The editorial staff was responsive, professional, and always willing to help. They provided valuable guidance on formatting, structure, and ethical considerations, making the submission process seamless. Moreover, they kept me informed about the status of my manuscript and provided timely updates, which made the process less stressful. The journal Brain and Neurological Disorders is of the highest quality, with a strong focus on publishing cutting-edge research in the field of neurology. The articles published in this journal are well-researched, rigorously peer-reviewed, and written by experts in the field. The journal maintains high standards, ensuring that readers are provided with the most up-to-date and reliable information on brain and neurological disorders. In conclusion, I had a wonderful experience publishing in Brain and Neurological Disorders. The peer review process was thorough, the editorial office provided exceptional support, and the journal's quality is second to none. I would highly recommend this journal to any researcher working in the field of neurology and brain disorders.

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Dr Shiming Tang

Dear Agrippa Hilda, Journal of Neuroscience and Neurological Surgery, Editorial Coordinator, I trust this message finds you well. I want to extend my appreciation for considering my article for publication in your esteemed journal. I am pleased to provide a testimonial regarding the peer review process and the support received from your editorial office. The peer review process for my paper was carried out in a highly professional and thorough manner. The feedback and comments provided by the authors were constructive and very useful in improving the quality of the manuscript. This rigorous assessment process undoubtedly contributes to the high standards maintained by your journal.

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Raed Mualem

International Journal of Clinical Case Reports and Reviews. I strongly recommend to consider submitting your work to this high-quality journal. The support and availability of the Editorial staff is outstanding and the review process was both efficient and rigorous.

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Andreas Filippaios

Thank you very much for publishing my Research Article titled “Comparing Treatment Outcome Of Allergic Rhinitis Patients After Using Fluticasone Nasal Spray And Nasal Douching" in the Journal of Clinical Otorhinolaryngology. As Medical Professionals we are immensely benefited from study of various informative Articles and Papers published in this high quality Journal. I look forward to enriching my knowledge by regular study of the Journal and contribute my future work in the field of ENT through the Journal for use by the medical fraternity. The support from the Editorial office was excellent and very prompt. I also welcome the comments received from the readers of my Research Article.

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Dr Suramya Dhamija

Dear Erica Kelsey, Editorial Coordinator of Cancer Research and Cellular Therapeutics Our team is very satisfied with the processing of our paper by your journal. That was fast, efficient, rigorous, but without unnecessary complications. We appreciated the very short time between the submission of the paper and its publication on line on your site.

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Bruno Chauffert

I am very glad to say that the peer review process is very successful and fast and support from the Editorial Office. Therefore, I would like to continue our scientific relationship for a long time. And I especially thank you for your kindly attention towards my article. Have a good day!

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Baheci Selen

"We recently published an article entitled “Influence of beta-Cyclodextrins upon the Degradation of Carbofuran Derivatives under Alkaline Conditions" in the Journal of “Pesticides and Biofertilizers” to show that the cyclodextrins protect the carbamates increasing their half-life time in the presence of basic conditions This will be very helpful to understand carbofuran behaviour in the analytical, agro-environmental and food areas. We greatly appreciated the interaction with the editor and the editorial team; we were particularly well accompanied during the course of the revision process, since all various steps towards publication were short and without delay".

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Jesus Simal-Gandara

I would like to express my gratitude towards you process of article review and submission. I found this to be very fair and expedient. Your follow up has been excellent. I have many publications in national and international journal and your process has been one of the best so far. Keep up the great work.

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Douglas Miyazaki

We are grateful for this opportunity to provide a glowing recommendation to the Journal of Psychiatry and Psychotherapy. We found that the editorial team were very supportive, helpful, kept us abreast of timelines and over all very professional in nature. The peer review process was rigorous, efficient and constructive that really enhanced our article submission. The experience with this journal remains one of our best ever and we look forward to providing future submissions in the near future.

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Dr Griffith

I am very pleased to serve as EBM of the journal, I hope many years of my experience in stem cells can help the journal from one way or another. As we know, stem cells hold great potential for regenerative medicine, which are mostly used to promote the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives. I think Stem Cell Research and Therapeutics International is a great platform to publish and share the understanding towards the biology and translational or clinical application of stem cells.

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Dr Tong Ming Liu

I would like to give my testimony in the support I have got by the peer review process and to support the editorial office where they were of asset to support young author like me to be encouraged to publish their work in your respected journal and globalize and share knowledge across the globe. I really give my great gratitude to your journal and the peer review including the editorial office.

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Husain Taha Radhi

I am delighted to publish our manuscript entitled "A Perspective on Cocaine Induced Stroke - Its Mechanisms and Management" in the Journal of Neuroscience and Neurological Surgery. The peer review process, support from the editorial office, and quality of the journal are excellent. The manuscripts published are of high quality and of excellent scientific value. I recommend this journal very much to colleagues.

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S Munshi

Dr.Tania Muñoz, My experience as researcher and author of a review article in The Journal Clinical Cardiology and Interventions has been very enriching and stimulating. The editorial team is excellent, performs its work with absolute responsibility and delivery. They are proactive, dynamic and receptive to all proposals. Supporting at all times the vast universe of authors who choose them as an option for publication. The team of review specialists, members of the editorial board, are brilliant professionals, with remarkable performance in medical research and scientific methodology. Together they form a frontline team that consolidates the JCCI as a magnificent option for the publication and review of high-level medical articles and broad collective interest. I am honored to be able to share my review article and open to receive all your comments.

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Tania Munoz

“The peer review process of JPMHC is quick and effective. Authors are benefited by good and professional reviewers with huge experience in the field of psychology and mental health. The support from the editorial office is very professional. People to contact to are friendly and happy to help and assist any query authors might have. Quality of the Journal is scientific and publishes ground-breaking research on mental health that is useful for other professionals in the field”.

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George Varvatsoulias

Dear editorial department: On behalf of our team, I hereby certify the reliability and superiority of the International Journal of Clinical Case Reports and Reviews in the peer review process, editorial support, and journal quality. Firstly, the peer review process of the International Journal of Clinical Case Reports and Reviews is rigorous, fair, transparent, fast, and of high quality. The editorial department invites experts from relevant fields as anonymous reviewers to review all submitted manuscripts. These experts have rich academic backgrounds and experience, and can accurately evaluate the academic quality, originality, and suitability of manuscripts. The editorial department is committed to ensuring the rigor of the peer review process, while also making every effort to ensure a fast review cycle to meet the needs of authors and the academic community. Secondly, the editorial team of the International Journal of Clinical Case Reports and Reviews is composed of a group of senior scholars and professionals with rich experience and professional knowledge in related fields. The editorial department is committed to assisting authors in improving their manuscripts, ensuring their academic accuracy, clarity, and completeness. Editors actively collaborate with authors, providing useful suggestions and feedback to promote the improvement and development of the manuscript. We believe that the support of the editorial department is one of the key factors in ensuring the quality of the journal. Finally, the International Journal of Clinical Case Reports and Reviews is renowned for its high- quality articles and strict academic standards. The editorial department is committed to publishing innovative and academically valuable research results to promote the development and progress of related fields. The International Journal of Clinical Case Reports and Reviews is reasonably priced and ensures excellent service and quality ratio, allowing authors to obtain high-level academic publishing opportunities in an affordable manner. I hereby solemnly declare that the International Journal of Clinical Case Reports and Reviews has a high level of credibility and superiority in terms of peer review process, editorial support, reasonable fees, and journal quality. Sincerely, Rui Tao.

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Rui Tao

Clinical Cardiology and Cardiovascular Interventions I testity the covering of the peer review process, support from the editorial office, and quality of the journal.

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Khurram Arshad